Smoke inhalation and burn injury (SIBI) induces acute lung injury (ALI) via the generation of toxic free radical and oxidant species that, in turn, trigger DNA single strand breaks (SSB) throughout the pulmonary parenchyma. When this genomic injury is then recognized by the nuclear DNA repair enzyme poly(ADP-ribose) polymerase (PARP), NAD is cleaved to generate the product poly(ADP-ribose). Hyperactivation of PARP depletes NAD stores, resulting in the loss of oxidative phosphorylation and interference with ATP synthesis. In severe instances, the ultimate exhaustion of high energy phosphates (ATP) triggers cellular necrosis and the functional loss of the alveolocapillary membrane. To address this unmet clinical need and potential orphan drug market, we are developing a cell-permeable thioredoxin mimetic (R-901), a thiol-rich tripeptide that is closely analogous to the native thioredoxin (TRX) motif. R-901 exhibits in vitro potency 450- and 50-fold > than N-acetylcysteine (NAC) and ascorbic acid, respectively, and in vivo potency substantialy greater than NAC. Therapeutic administration of R-901 has been shown in a murine model of pulmonary inflammation (induced by ovalbumin sensitization and re-challenge) to reduce histologic injury, diminish neutrophil (PMN) infiltration, attenuate tissue oxidation, block pro-inflammatory cytokine expression and nuclear translocation of NF-?B, diminish the degradation of the anti-inflammatory cytoplasmic protein I?B, and restore the balance of reduced and oxidized forms of glutathione. In a murine LD100 model of severe redox stress induced by acute chlorine gas inhalation, post-insult administration of R-901 reduced pulmonary PMN infiltration by 50%. The Specific Aim of this proposal is to compare the potency of R-901 and NAC in an ovine model of combined SIBI-ALI. We will carry out a prospective, placebo-controlled, randomized, double-blinded, study in which respiratory insufficiency is induced in mechanically-ventilated Merino sheep by acute SIBI. Resuscitation with R-901, NAC, or vehicle control will be initiated 1 h after thermal insult and continued for 48 h, a timepoint characterized by massive respiratory failure. We expect that R-901 will demonstrate statistically significant superiority relative to NAC as noted for the following indices: 1) Reduced lung inflammation and injury, as manifested in lung wet/dry ratio, lymphatic drainage, lipid peroxidation, airway obstruction, histologic scoring, PMN infiltration, ONOO- (3-nitrotyrosine, 3-NT) formation, and poly(ADP-ribose) (PAR) formation; 2) Improved hemodynamics and airway pressures, as revealed by reductions in pulmonary vascular resistance (PVR), mean pulmonary arterial pressure (MPAP), and peak inspiratory pressure (PIP), without impacting systemic vascular resistance (SVR), mean peripheral arterial pressure (MAP), and cardiac index (CI); and 3) Reduced pulmonary shunt, as indicated by diminished arterioalveolar (Aa) gradient and an increased ratio of arterial O2 tension to inspired O2 concentration (PaO2/FiO2). These benefits, taken together, will justify continued development of R-901 for treatment of acute SIBI. PUBLIC HEALTH RELEVANCE: Smoke inhalation with associated burn injury is a leading cause of respiratory failure. At present, there is no approved therapy for this condition and mortality remains high. We are developing a novel drug that targets the basic mechanisms of lung injury in this condition and will test this agent in a clinically-relevant large animal model f trauma.